Method and compositions for the synthesis of BCH-189 and related compounds

ABSTRACT

The present invention relates to a method of preparing BCH-189 and various analogs of BCH-189 from inexpensive precursors with the option of introducing functionality as needed. This synthetic route allows the stereoselective preparation of the biologically active isomer of these compounds, β-BCH-189 and related compounds. Furthermore, the steochemistry at the nucleoside 4&#39; position can be controlled to produce enantiomerically-enriched β-BCH-189 and its analogs.

The invention described herein was made with Government support undergrant no. 5-21935 awarded by the National Institutes of Health. TheGovernment has certain rights in this invention.

This application is a divisional of 07/473,318, filed Feb. 1, 1990, nowU.S. Pat. No. 5,204,406.

BACKGROUND OF THE INVENTION

The present invention relates to methods and compositions for preparingantiviral nucleoside analogs, particularly BCH-189(2',3'-dideoxy-3'-thia-cytidine). More particularly, the inventionrelates to the selective synthesis of the β-isomer of BCH-189 andrelated compounds as well as the selective synthesis ofenantiomerically-enriched BCH-189 and related compounds.

In 1981, documentation began on the disease that became known asAcquired Immune Deficiency Syndrome (AIDS), as well as its forerunnerAIDS Related Complex (ARC). In 1983, the cause of the disease AIDS wasestablished as a virus named the Human Immunodeficiency Virus type 1(HIV-1). Usually, a person infected with the virus will eventuallydevelop AIDS; in all known cases of AIDS the final outcome has alwaysbeen death.

The disease AIDS is the end result of an HIV-1 virus following its owncomplex life cycle. The virion life cycle begins with the virionattaching itself to the host human T-4 lymphocyte immune cell throughthe bonding of a glycoprotein on the surface of the virion's protectivecoat with the CD4 glycoprotein on the lymphocyte cell. Once attached,the virion sheds its glycoprotein coat, penetrates into the membrane ofthe host cell, and uncoats its RNA. The virion enzyme, reversetranscriptase, directs the process of transcribing the RNA into singlestranded DNA. The viral RNA is degraded and a second DNA strand iscreated. The now double-stranded DNA is integrated into the human cell'sgenes and those genes are used for cell reproduction.

At this point, the human cell carries out its reproductive process byusing its own RNA polymerase to transcribe the integrated DNA into viralRNA. The viral RNA is translated into glycoproteins, structuralproteins, and viral enzymes, which assemble with the viral RNA intact.When the host cell finishes the reproductive step, a new virion cell,not a T-4 lymphocyte, buds forth. The number of HIV-1 virus cells thusgrows while the number of T-4 lymphocytes decline.

The typical human immune system response, killing the invading virion,is taxed because a large portion of the virion's life cycle is spent ina latent state within the immune cell. In addition, viral reversetranscriptase, the enzyme used in making a new virion cell, is not veryspecific, and causes transcription mistakes that result in continuallychanged glycoproteins on the surface of the viral protective coat. Thislack of specificity decreases the immune system's effectiveness becauseantibodies specifically produced against one glycoprotein may be uselessagainst another, hence reducing the number of antibodies available tofight the virus. The virus continues to grow while the immune responsesystem continues to weaken. Eventually, the HIV largely holds free reignover the body's immune system, allowing opportunistic infections to setin and ensuring that, without the administration of antiviral agentsand/or immunomodulators, death will results.

There are three critical points in the virus's life cycle which havebeen identified as targets for antiviral drugs: (1) the initialattachment of the virion to the T-4 lymphocyte, or macrophage, site, (2)the transcription of viral RNA to viral DNA, and (3) the assemblage ofthe new virion cell during reproduction.

Inhibition of the virus at the second critical point, the viral RNA tovital DNA transcription process, has provided the bulk of the therapiesused in treating AIDS. This transcription must occur for the virion toreproduce because the virion's genes are encoded in RNA; the host cellreads only DNA. By introducing drugs that block the reversetranscriptase from completing the formation of viral DNA, HIV-1replication can be stopped.

Nucleoside analogs, such as 3'-azido-3'-deoxythymidine (AZT),2',3'-dideoxycytidine (DDC), 2',3'-dideoxythymidinene (D4T),2',3'-dideoxyinosine (DDI), and various fluoro-derivatives of thesenucleosides are relatively effective in halting HIV replication at thereverse transcriptase stage. Another promising reverse transcriptaseinhibitor is 2',3'-dideoxy-3'-thia-cytidine (BCH-189), which contains anoxathiolane ring substituting for the sugar moiety in the nucleoside.

AZT is a successful anti-HIV drug because it sabotages the formation ofviral DNA inside the host T-4 lymphocyte cell. When AZT enters the cell,cellular kinases activate AZT by phosphorylation to AZT triphosphate.AZT triphosphate then competes with natural thymidine nucleosides forthe receptor site of HIV reverse transcriptase enzyme. The naturalnucleoside possesses two reactive ends, the first for attachment to theprevious nucleoside and the second for linking to the next nucleoside.The AZT molecule has only the first reactive end; once inside the HIVenzyme site, the AZT azide group terminates viral DNA formation becausethe azide cannot make the 3',5'-phosphodiester with the ribose moiety ofthe following nucleoside.

AZT's clinical benefits include increased longevity, reduced frequencyand severity of opportunistic infections, and increased peripheral CD4lymphocyte count. Immunosorbent assays for viral p24, an antigen used totrack HIV-1 activity, show a significant decrease with use of AZT.However, AZT's benefits must be weighed against the severe adversereactions of bone marrow suppression, nausea, myalgia, insomnia, severeheadaches, anemia, peripheral neuropathy, and seizures. Furthermore,these adverse side effects occur immediately after treatment beginswhereas a minimum of six weeks of therapy is necessary to realize AZT'sbenefits.

Both DDC and D4T are potent inhibitors of HIV replication withactivities comparable (D4T) or superior (DDC) to AZT. However, both DDCand D4T are converted to their 5' triphosphates less efficiently thantheir natural analogs and are resistent to deaminases andphosphorylases. Clinically, both compounds are toxic. Currently, DDI isused in conjunction with AZT to treat AIDS. However, DDI's side effectsinclude sporadic pancreatis and peripheral neuropathy. Initial tests on3'-fluoro-2'-3'-dideoxythymidine show that its anti-viral activity iscomparable to that of AZT.

Recent tests on BCH-189 have shown that it possesses anti-HIV activitysimilar to AZT and DDC, but without the cell toxicity which causes thedebilitating side effects of AZT and DDC. A sufficient quantity ofBCH-189 is needed to allow clinical testing and treatment using thedrug.

The commonly-used chemical approaches for synthesizing nucleosides ornucleoside analogs can be classified into two broad categories: (1)those which modify intact nuceosides by altering the carbohydrate, thebase, or both and (2) those which modify carbohydrates and incorporatethe base, or its synthetic precursor, at a suitable stage in thesynthesis. Because BCH-189 substitutes a sulfur atom for a carbon atomin the carbohydrate ring, the second approach is more feasible. The mostimportant factor in this latter strategy involves delivering the basefrom the β-face of the carbohydrate ring in the glycosylation reactionbecause only the β-isomers exhibit useful biological activity.

It is well known in the art that the stereoselective introduction ofbases to the anomeric centers of carbohydrates can be controlled bycapitalizing on the neighboring group participation of a 2-substituenton the carbohydrate ring (Chem. Ber. 114:1234 (1981)). However, BCH-189and its analogs do not possess a 2-substitutent and, therefore, cannotutilize this procedure unless additional steps to introduce a functionalgroup that is both directing and disposable are incorporated into thesynthesis. These added steps would lower the overall efficiency of thesynthesis.

It is also well known in the art that "considerable amounts of theundesired α-nucleosides are always formed during the synthesis of2'-deoxyribosides" (Chem. Ber. 114:1234, 1244 (1981)). Furthermore, thisreference teaches that the use of simple Friedel-Crafts catalysts likeSnCl₄ in nucleoside syntheses produces undesirable emulsions upon theworkup of the reaction mixture, generates complex mixtures of the α andβ-isomers, and leads to stable p-complexes between the SnCl₄ and themore basic silyated heterocycles such as silyated cytosine. Thesecomplexes lead to longer reaction times, lower yields, and production ofthe undesired unnatural N-3-nucleosides. Thus, the prior art teaches theuse of trimethysilyl triflate or trimethylsilyl perchlorate as acatalyst during the coupling of pyrimidine bases with a carbohydratering to achieve high yields of the biologically active β-isomers.However, the use of these catalysts to synthesize BCH-189 or BCH-189analogs does not produce the β-isomer preferentially; these reactionsresult in approximately a 50:50 ratio of the isomers.

Thus, there exists a need for an efficient synthetic route to BCH-189and its analogs. There also exists a need for a stereoselectivesynthetic route to the biologically active isomer of these compounds,β-BCH-189 and related β-analogs. Furthermore, there exists a need for astereoselective synthetic route to enantiomerically-enriched β-BCH-189because the other enantiomer is inactive and, therefore, represents a50% impurity.

SUMMARY OF THE INVENTION

The present invention relates to the discovery of a surprisinglyefficient synthetic route to BCH-189 and various analogs of BCH-189 frominexpensive precursors with the option of introducing functionality asneeded. This synthetic route allows the stereoselective preparation ofthe biologically active isomer of these compounds, β-BCH-189 and relatedcompounds. Furthermore, the steochemistry at the nucleoside 4' positioncan be controlled to produce enantiomerically-enriched β-BCH-189 and itsanalogs.

The term "BCH-189 analogs" is meant to refer to nucleosides that areformed from pyrimidine bases substituted at the 5 position that arecoupled to substituted 1,3-oxathiolanes.

The method of the present invention includes ozonizing an allyl ether orester having the formula CH₂ ═CH-CH₂ -OR, in which R is a protectinggroup, such as an alkyl, silyl, or acyl group, to form a glycoaldehydehaving the formula OHC-CH₂ -OR; adding thioglycolic acid to theglycoaldehyde to form a lactone of the formula2-(R-oxy)-methyl-5-oxo-1,3-oxathiolane; converting the lactone to itscorresponding carboxylate at the 5 position of the oxathiolane ring;coupling the acetate with a silyated pyrimidine base in the presence ofSnCl₄ to form the β-isomer of a 5'-(R-oxy)-2',3'-dideoxy-3'-thia-nucleoside analog; and replacing the R protecting group with a hydrogento form BCH-189 or an analog of BCH-189.

The invention can be used to produce BCH-189 or BCH-189 analogs that areenantiomerically-enriched at the 4' position by selecting an appropriateR protecting group to allow stereoselective selection by an enzyme. Forinstance, the R protecting group can be chosen such that the substituentat the 2 position of the oxathiolane lactone is butyryloxy to permitstereoselective enzymatic hydrolysis by pig liver esterase. Theresulting optically active hydrolyzed lactone can then be converted toits corresponding diacetate and coupled with a silyated pyrimidine baseas above.

Accordingly, one of the objectives of this invention is to provide anefficient method for preparing the β-isomer of BCH-189 and analogs ofBCH-189 in high yields. Furthermore, it is an objective of thisinvention to provide a synthetic method to produce only one opticalisomer, rather than a racemic mixture, of BCH-189 and analogs ofBCH-189. A further object of this invention is to provide a syntheticroute to produce β-BCH-189 that is enantiomerically-enriched.

Additionally, an objective of this invention is to provide intermediatesfrom which BCH-189 or BCH-189 analogs can be synthesized of the formula2-(R-oxymethyl)-5-acyloxy-1,3-oxathiolane, wherein R is a protectinggroup, such as alkyl, silyl, or acyl, and a method of preparing thesecompounds. Furthermore, it is an object of this invention to provideenantiomerically-enriched 2-acetoxymethyl-5-acetoxy-1,3-oxathiolane and2-butoxymethyl-5-oxo-1,3-oxathiolane and methods of preparing thesecompounds.

Another objective of this invention is to provide intermediates fromwhich BCH-189 or BCH-189 analogs can be synthesized of the formula:##STR1## wherein R is a protecting group, such as alkyl, silyl, or acyl,and Y can be hydrogen, methyl, halo, alkyl, alkenyl, alkynyl,hydroxalkyl, carboxalkyl, thioalkyl, selenoalkyl, phenyl, cycloalkyl,cycloalkenyl, thioaryl, and selenoaryl, and methods of preparing thesecoumpounds.

Furthermore, this invention provides intermediates from which BCH-189 orBCH-189 analogs can be synthesized of the formula: ##STR2## wherein R isa protecting group, such as alkyl, silyl, or acyl, and Y can behydrogen, methyl, halo, alkyl, alkenyl, alkynyl, hydroxalkyl,carboxalkyl, thioalkyl, selenoalkyl, phenyl, cycloalkyl, cycloalkenyl,thioaryl, and selenoaryl, and methods of preparing these coumpounds.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates one embodiment of a synthesis of BCH-189 and BCH-189analogs according to the present invention;

FIG. 2 illustrates one embodiment of the synthesis of BCH-189 accordingto the present invention;

FIG. 3 illustrates one embodiment of the synthesis of 5-methylcytidineand thymidine derivatives of BCH-189 according to the present invention;and

FIG. 4 illustrates one embodiment-of the synthesis ofenantiomerically-enriched BCH-189 according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

BCH-189 is a compound of the formula: ##STR3##

The process of the present invention for preparing BCH-189 and BCH-189analogs is set forth in FIG. 1. An allyl ether or ester 1 is ozonized togive an aldehyde 2, which reacts with thioglycolic acid to give alactone 3. The lactone 3 is treated with a reducing agent, followed by acarboxylic anhydride, to produce the carboxylate 4. This carboxylate iscoupled with a silyated pyrimidine base in the presence of a Lewis acidthat can catalyze stereospecific coupling, such as SnCl₄, to yield theβ-isomer of the substituted nucleoside 5 in essentially a 100:0 ratio ofβ:α isomers. The substituted nucleoside 5 is deprotected to produceBCH-189 or BCH-189 analog 6.

This procedure can be tailored to produce BCH-189 or BCH-189 analogsthat are enantiomerically-enriched at the 4' position by selecting anappropriate R protecting group to allow stereoselective enzymatichydrolysis of 3 by an enzyme such as pig liver esterase, porcinepancreatic lipase, or subtilisin or other enzymes that hydrolyze 3 in astereoselective fashion. The resulting optically active 3 can beconverted to enantiomerically-enriched carboxylate 4 and coupled with asilyated pyrimidine base as above to produce enantiomerically-enrichedBCH-189 or BCH-189 analogs.

The protecting group R in 1 can be selected to provide protection forthe corresponding alcohol until the final step in the synthesis iscarried out (deprotection of 5 to form 6). Additionally, the protectinggroup can be selected, if desired, to provide an additional recognitionsite for an enzyme to be used later in an enantio-selective hydrolysisreaction. For example, the lower alkyl ester of the β-isomer of BCH-189can be resolved into its (+) and (-)-enantiomers by treatment with pigliver esterase, porcine pancreatic lipase, or subtilisin, by methodsdescribed in detail herein. Any group that functions in this manner maybe used. For instance, alkyl, silyl, and acyl protecting groups orgroups that possess substantially the same properties as these groupscan be used.

An alkyl protecting group, as used herein, means triphenylmethyl or analkyl group that possesses substantially the same protecting propertiesas triphenylmethyl. A silyl protecting group, as used herein, means atrialkylsilyl group having the formula: ##STR4## wherein R₁, R₂, and R₃may be lower-alkyl, e.g., methyl, ethyl, butyl, and alkyl possessing 5carbon atoms or less; or phenyl. Furthermore, R₁ may be identical to R₂; R₁, R₂, and R₃ may all be identical. Examples of silyl protectinggroups include, but are not limited to, trimethylsilyl andt-butyldiphenylsilyl.

An acyl group, as used herein to describe an acyl protecting group (asin 1) or to describe a carboxylate (as in 4), is a group having theformula: ##STR5## wherein R' is a lower alkyl, e.g., methyl, ethyl,butyl, and alkyl possessing 5 carbon atoms or less; substituted loweralkyl wherein the alkyl bears one, two, or more simple substituents,including, but not limited to, amino, carboxyl, hydroxy, phenyl,lower-alkoxy, e.g., methoxy and ethoxy; phenyl; substituted phenylwherein the phenyl bears one, two, or more simple substituents,including, but not limited to, lower alkyl, halo, e.g., chloro andbromo, sulfato, sulfonyloxy, carboxyl, carbo-lower-alkoxy, e.g.,carbomethoxy and carbethoxy, amino, mono- and di- lower alkylamino,e.g., methylamino, amido, hydroxy, lower alkoxy, e.g., methoxy andethoxy, lower-alkanoyloxy, e.g., acetoxy.

A silyated pyrimidine base, as used herein, means a compound having theformula: ##STR6## wherein X is either a trialkylsilyloxy or atrialkylsilylamino group, Z is a trialkylsilyl group, and Y is furtherdescribed below. A trialkylsilyl group, as used herein, means a grouphaving the formula: ##STR7## wherein R₁, R₂, and R₃ may be lower-alkyl,e.g., methyl, ethyl, butyl, and alkyl possessing 5 carbon atoms or less,or phenyl. Furthermore, R₁ may be identical to R₂ ; R₁, R₂, and R₃ mayall be identical. Examples of trialkylsilyl groups include, but are notlimited to, trimethylsilyl and t-butyldiphenylsilyl.

The silyated pyrimidine base may be substituted with various Ysubstituents, including, but not limited to, hydrogen, methyl, halo,alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyalkyl, thioalkyl,selenoalkyl, phenyl, cycloalkyl, cycloalkenyl, thioaryl, and selenoaryl,at position 5 of the silyated pyrimidine base (Y substituent in FIG. 1)to modify the properties, such as transport properties or the rate ofmetabolism, of the BCH-189 analog.

Illustrative examples of the synthesis of BCH-189 or BCH-189 analogsaccording to the present invention are given in FIGS. 2, 3, and 4 andthe following descriptions.

FIG. 2 shows the synthesis of BCH-189 starting with allyl alcohol 7. ANaH oil suspension (4.5 g, 60%, 110 mmol) was washed with THF twice (100ml×2) and the resulting solid suspended in THF (300 ml). The suspensionwas cooled to 0° C., allyl alcohol Z (6.8 ml, 100 mmol) was addeddropwise, and the mixture was stirred for 30 minutes at 0° C.t-Butyl-diphenylsilyl chloride (25.8 ml, 100.8 mmol) was added dropwiseat 0° C. and the reaction mixture was stirred for 1 hour at 0° C. Thesolution was quenched with water (100 ml), and extracted with diethylether (200 ml×2). The combined extracts were washed with water, driedover MgSO₄, filtered, concentrated, and the residue distilled undervacuum (90°-100° C. at 0.5-0.6 mm Hg) to give a colorless liquid 8 (28g., 94 mmol, 94%). (¹ H NMR: 7.70-7.35 (10H, m, aromatic-H); 5.93 (1H,m, H₂); 5.37 (1H, dt, H₁) J=1.4 and 14.4 Hz; 5.07 (1H, dt, H₁) J=1.4 and8.7 Hz; 4.21 (2H, m, H₃); 1.07 (9H, s, t-Bu))

The silyl allyl ether 8 (15.5 g, 52.3 mmol) was dissolved in CH₂ Cl₂(400 ml), and ozonized at -78° C. Upon completion of ozonolysis, DMS (15ml, 204 mmol, 3.9 eq) was added at -78° C. and the mixture was warmed toroom temperature and stirred overnight. The solution was washed withwater (100 ml×2),dried over MgSO₄, filtered, concentrated, and distilledunder vacuum (100°-110° C. at 0.5-0.6 mm Hg) to give a colorless liquid9 (15.0 g, 50.3 mmol, 96%). (¹ H NMR: 9.74 (1H, s, H-CO); 7.70-7.35(10H, m, aromatic-H); 4.21 (2H, s, --CH₂); 1.22 (9H, s, t-Bu))

Silayted glycoaldehyde 9 (15.0 g, 50.3 mmol) was dissolved in toluene(200 ml) and thioglycolic acid (3.50 ml, 50.3 mmol) was added all atonce. The solution was refluxed for 2 hours while the resulting waterwas removed with a Dean-Stark trap. The solution was cooled to roomtemperature and washed with saturated NaHCO₃ solution and the aqueouswashings were extracted with diethyl ether (200 ml×2). The combinedextracts were washed with water (100 ml×2), dried over MgSO₄, filtered,and concentrated to give a colorless oil 10 (16.5 g, 44.3 mmol, 88%),which gradually solidified under vacuum. Recrystallization from hexaneafforded a white solid 10. (15.8 g, 84%). (¹ H NMR: 7.72-7.38 (10H, m,aromatic-H); 5.53 (1H, t, H₂) J=2.7 Hz; 3.93 (1H, dd, --CH₂ O) J=9.3 Hz;3.81 (1H, d, 1H₄) J=13.8 Hz; 3.79 (1H, dd, --CH₂ O); 3.58 (1H, d, 1H₄);1.02 (9H, s, t-Bu))

2-(t-Butyl-diphenylsilyloxy)-methyl-5-oxo-1,2-oxathiolane 10 (5.0 g,13.42 mmol) was dissolved in toluene (150 ml) and the solution wascooled to -78° C. Dibal-H solution (14 ml, 1.0 M in hexanes, 14 mmol)was added dropwise, while the inside temperature was kept below -70° C.all the time. After the completion of the addition, the mixture wasstirred for 30 minutes at -78° C. Acetic anhydride (5 ml, 53 mmol) wasadded and the mixture was warmed to room temperature and stirredovernight. Water (5 ml) was added to the mixture and the resultingmixture was stirred for 1 hour at room temperature. The mixture wasdiluted with diethyl ether (300 ml), MgSO₄ (40 g) was added, and themixture was stirred vigorously for 1 hour at room temperature. Themixture was filtered, concentrated, and the residue flashchromatographed with 20% EtOAc in hexanes to give a colorless liquid 11(3.60 g, 8.64 mmol, 64%), which was a 6:1 mixture of anomers. (¹ H NMRof the major isomer: 7.70-7.35 (10H, m, aromatic-H); 6.63 (1H, d, H₅)J=4.4 Hz; 5.47 (1H, t, H₂); 4.20-3.60 (2H, m, --CH₂ O); 3.27 (1H, dd,1H₄) J=4.4 and 11.4 Hz; 3.09 (1H, d, 1H₄) J=11.4 Hz; 2.02 (3H, s, CH₃CO); 1.05 (9H, s, t-Bu); ¹ H NMR of the minor isomer: 7.70-7.35 (10H, m,aromatic-H); 6.55 (1H, d, H₅) J=3.9 Hz; 5.45 (1H, t, H₂); 4.20-3.60 (2H,m, --CH₂ O); 3.25 (1H, dd, 1H₄) J=3.9 and 11.4 Hz; 3.11 (1H, d, 1H₄)J=11.4 Hz; 2.04 (3H, s, CH₃ CO); 1.04 (9H, s, t-Bu))

2-(t-Butyl-diphenylsilyloxy)-methyl-5-acetoxy-1,3-oxathiolane 11 (0.28g, 0.67 mmol) was dissolved in 1,2-dichloroethane (20 ml), and silylatedcytosine 12 (0.20 g, 0.78 mmol) was added at once at room temperature.The mixture was stirred for 10 minutes and to it was added SnCl₄solution (0.80 ml, 1.0 M solution in CH₂ Cl₂, 0.80 mmol) dropwise atroom temperature. Additional cytosine 12 (0.10 g, 0.39 mmol) and SnCl₄solution (0.60 ml) were added in a same manner 1 hour later. Aftercompletion of the reaction in 2 hours, the solution was concentrated,and the residue was triturated with triethylamine (2 ml ) and subjectedto flash chromatography (first with neat EtOAc and then 20% ethanol inEtOAc) to give a tan solid 13 (100% β configuration) (0.25 g, 0.54 mmol,80%). (¹ H NMR (DMSO-d⁶): 7.75 (1H, d, H₆) J=7.5 Hz; 7.65-7.35 (10H, m,aromatic-H); 7.21 and 7.14 (2H, broad, --NH₂); 6.19 (1H, t, H₅,); 5.57(1H, d, H₅); 5.25 (1H, t, H₂,); 3.97 (1H; dd, --CH₂ O) J=3.9 and 11.1Hz; 3.87 (1H, dd, --CH₂ O); 3.41 (1H, dd, 1H₄,) J=4.5 and 11.7 Hz; 3.03(1H, dd, 1H₄,) J=?; 0.97 (9H, s, t-Bu))

Silyether 13 (0.23 g, 0.49 mmol) was dissolved in THF (30 ml), and to itwas added n-Bu₄ NF solution (0.50 ml, 1.0 M solution in THF, 0.50 mmol)dropwise at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up withethanol/triethylamine (2 ml/1 ml), and subjected to flash chromatography(first with EtOAc, then 20% ethanol in EtOAc) to afford a white solid 14in 100% anomeric purity (BCH-189; 0.11 g, 0.48 mmol, 98%), which wasfurther recrystallized from ethanol/CHCl₃ /Hexanes mixture. (¹ H NMR(DMSO-d₆): 7.91 (1H, d, H₆) J=7.6 Hz; 7.76 and 7.45 (2H, broad, --NH₂);6.19 (1H, t, H₅,); 5.80 (1H, d, H₅) J=7.6 Hz; 5.34 (1H, broad, --OH);5.17 (1H, t, H₂,); 3.74 (2H, m, --CH₂ O); 3.42 (1H, dd, 1H₄,) J=5.6 and11.5 Hz; 3.09 (1H, dd, 1H₄,) J=4.5 and 11.5 Hz)

BCH-189 and its analogs can also be synthesized by coupling a silylateduracil derivative with 11. Silylated uracil derivative 15 (1.80 g, 7.02mmol) was coupled with 11 (1.72 g, 4.13 mmol) in 1,2-dichloroethane (50ml) in the presence of SnCl₄ (5.0 ml) as described above in the thepreparation of the cytosine derivative 13. The reaction was completeafter 5 hours. Flash chromatography, first with 40% EtOAc in hexane andthen EtOAc, afforded a white foam 16 (1.60 g, 3.43 mmol, 83%). (¹ H NMR:9.39 (1H, broad, --NH) 7.90 (1H, d, H₆) J=7.9 Hz; 7.75-7.35 (10H, m,aromatic-H); 6.33 (1H, dd, H₅,); 5.51 (1H, d, H₅) J=7.9 Hz; 5.23 (1H, t,H₂,); 4.11 (1H, dd, --CH₂ O) J=3.2 and 11.7 Hz; 3.93 (1H, dd, --CH₂ O);3.48 (1H, dd, 1H₄,) J=5.4 and 12.2 Hz; 3.13 (1H, dd, 1H₄,) J=3.2 and12.2 Hz)

The uracil derivative 16 can be converted to the cytosine derivative 13.The uracil derivative 16 (0.20 g, 0.43 mmol) was dissolved in a mixtureof pyridine/dichloroethane (2 ml/10 ml), and the solution cooled to 0°C. Triflic anhydride (72 μl, 0.43 mmol) was added dropwise at 0° C. andthe mixture was warmed to room temperature and stirred for 1 hour.Additional triflic anhydride (0.50 μl, 0.30 mmol) was added and themixture stirred for 1 hour. TLC showed no mobility with EtOAc. Thereaction mixture was then decannulated into a NH₃ -saturated methanolsolution (30 ml) and the mixture was stirred for 12 hours at roomtemperature. The solution was concentrated, and the residue subjected toflash chromatography to give a tanned foam 13. (0.18 g, 0.39 mmol, 91%),which was identical with the compound obtained from the cytosinecoupling reaction.

FIG. 3 illustrates the synthesis of 5-methylcytidine and thymidinederivatives of BCH-189. The acetate 11 (0.93 g, 2.23 mmol) in1,2-dichloroethane (50 ml), was reacted with the silylated thyminederivative 17 (1.0 g, 3.70 mmol), and SnCl₄ solution (4.0 ml) in amanner similar to that described for the preparation of cytosinederivative 13. (¹ H NMR: 8.10 (1H, broad, NH); 7.75-7.30 (11H, m, 10Aromatic H's and 1H₆); 6.32 (1H, t, H₁,) J=5.4 Hz; 5.25 (1H, t, H₄,)J=4.2 Hz; 4.01 (1H, dd, 1H₅,) J=3.9 and 11.4 Hz; 3.93 (1H, dd, 1H₅,)J=4.5 and 11.4 Hz; 3.41 (1H, dd, 1H₂,) J=5.4 and 11.7 Hz; 3.04 (1H, dd,1H₂,) J=5.7 and 11.7 Hz; 1.75 (3H, s, CH₃); 1.07 (9H, s, t-Bu))

The thymine derivative 18 (0.20 g, 0.42 mmol) was dissolved in a mixtureof pyridine/dichloroethane (2 ml/10 ml), and the solution cooled to 0°C. To it was added triflic anhydride (100 μl, 0.60 mmol) dropwise at 0°C., and the mixture was allowed, with continuous stirring, to warm toroom temperature. After reaching room temperature, it was stirred for 1hour. TLC showed no mobility with EtOAc. The reaction mixture was thendecannulated into the NH₃ -saturated methanol solution (20 ml), and themixture stirred for 12 hours at room temperature. The solution wasconcentrated, and the residue was subjected to flash chromatograhy togive a tanned foam 19 (0.18 g, 0.38 mmol, 90%). (¹ H NMR: 7.70-7.30(12H, m, 10 Aromatic H's, 1NH and H₆); 6.60 (1H, broad, 1NH); 6.34 (1H,t, H₁,) J=4.5 Hz; 5.25 (1H, t, H₄,) J=3.6 Hz; 4.08 (1H, dd, 1Hs,) J=3.6and 11.4 Hz; 3.96 (1H, dd, 1H₅,) J=3.6 and 11.4 Hz; 3.52 (1H, dd, 1H₂,)J=5.4 and 12.3 Hz; 3.09 (1H, dd, 1H₂,) J=3.9 and 12.3 Hz; 1.72 (3H, s,CH₃); 1.07 (9H, s, t-Bu))

Silylether 19 (0.18 g, 0.38 mmol) was dissolved in THF (20 ml), and ann-Bu₄ NF solution (0.50 ml, 1.0 M solution in THF, 0.50 mmol) was added,dropwise, at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up withethanol/triethylamine (2 ml/1 ml), and subjected to flash chromatography(first with EtOAc, then 20% ethanol in EtOAc) to afford a white solid 20(0.09 g, 0.37 mmol, 97%), which was futher recrystallized fromethanol/CHCl₃ /Hexanes mixture to afford 82 mg of pure compound (89%).(¹ H NMR: (in d⁶ -DMSO): 7.70 (1H, s, H₆); 7.48 and 7.10 (2H, broad,NH₂); 6.19 (1H, t, H₁,) J=6.5 Hz; 5.31 (1H, t, OH); 5.16 (1H, t, 1H₄,)J=5.4 Hz; 3.72 (2H, m, 2H₅,) 3.36 (1H, dd, 1H₂,) J=6.5 and 14.0 Hz; 3.05(1H, dd, 1H₂,) J=6.5 and 14.0 Hz; 1.85 (3H, s, CH₃))

Silylether 18 (0.70 g, 1.46 mmol) was dissolved in THF (50 ml), and ann-Bu₄ NF solution (2 ml, 1.0 M solution in THF, 2 mmol) was added,dropwise, at room temperature. The mixture was stirred for 1 hour andconcentrated under vacuum. The residue was taken up withethanol/triethylamine (2 ml/1 ml), and subjected to flash chromatographyto afford a white solid 21 (0.33 g, 1.35 mmol, 92%). (¹ H NMR: (in d₆-Acetone): 9.98 (1H, broad, NH); 7.76 (1H, d, H₆) J=1.2 Hz; 6.25 (1H, t,H₄,) J=5.7 Hz; 5.24 (1H, t, H₁,) J=4.2 Hz; 4.39 (1H, t, OH) J=5.7 Hz;3.85 (1H, dd, 2H₅,) J=4.2 and 5.7 Hz; 3.41 (1H, dd, 1H₂,) J=5.7 and 12.0Hz; 3.19 (1H, dd, 1H₂,) J=5.4 and 12.0 Hz; 1.80 (3H, s, CH₃))

FIG. 4 illustrates the synthesis of enantiomerically-enriched BCH-189and its analogs. Allyl butyrate 22 (19.0 g, 148 mmol) was dissolved inCH₂ Cl₂ (400 ml), and ozonized at -78° C. Upon completion of ozonolysis,dimethyl sulfide (20 ml, 270 mmol, 1.8 eq) was added at -78° C. and themixture was warmed to room temperature and stirred overnight. Thesolution was washed with water (100 ml×2), dried over MgSO₄, filtered,concentrated, and distilled under vacuum (70° 80° C. at 0.5-0.6 mm Hg)to give a colorless liquid 23 (17.0 g, 131 mmol, 88%). (¹ H NMR: 9.59(1H, s, H-CO); 4.66 (2H, s, --CH₂ O); 2.42 (2H, t, CH₂ CO) J=7.2 Hz;1.71 (2H, sex, --CH₂); 0.97 (3H, t, CH₃) J=7.2 Hz) (IR (neat): 2990,2960, 2900, 1750, 1740, 1460, 1420, 1390, 1280, 1190, 1110, 1060, 1020,990, 880, 800, 760)

Butyryloxyacetaldehyde 23 (15.0 g, 115 mmol) was dissolved in toluene(200 ml) and mixed with thioglycolic acid (8.0 ml, 115 mmol). Thesolution was refluxed for 5 hours while the resulting water was removedwith a Dean-Stark trap. The solution was cooled to room temperature andwas transferred to a 500 ml separatory funnel. The solution was thenwashed with saturated NaHCO₃ solution. These aqueous washing wereextracted with diethyl ether (200 ml×2) to recuperate any crude productfrom the aqueous layer. The ether extracts were added to the toluenelayer and the resulting mixture was washed with water (100 ml×2), driedover MgSO₄, filtered, concentrated, and distilled under vacuum (70°-80°C. at 0.5-0.6 mm Hg) to give a colorless oil 24 (19 g, 93 mmol, 81%). (¹H NMR: 5.65 (1H, dd, H₅) J=5.0 and 1.4 Hz; 4.35 (1H, dd, --CH₂ O) J=3.2and 12.2 Hz; 4.29 (1H, dd, --CH₂ O) J=5.7 and 12.2 Hz; 3.72 (1H, d,--CH₂ S) J=16.2 Hz; 3.64 (1H, d, --CH₂ S; 2.34 (2H, t, --CH₂ CO) J=7.2Hz; 1.66 (2H, sex, --CH₂); 0.95 (3H, t, CH₃) J=7.2 Hz) (IR (neat): 2980,2960, 2900, 1780, 1740, 1460, 1410, 1390, 1350, 1300, 1290, 1260, 1220,1170, 1110, 1080, 1070, 1000, 950, 910, 830, 820, 800, 760).

Pig liver esterase solution (90 μl) was added to a buffer solution (pH7, 100 ml) at room temperature, and the mixture stirred vigorously for 5minutes. The butyrate 24. (2.8 g, 13.7 mmol) was added, all at once, tothe esterase/buffer solution and the mixture was stirred vigorously atroom temperature for 2 hours. The reaction mixture was poured into aseparatory funnel. The reaction flask was washed with ether (10 ml) andthe washing was combined with the reaction mixture in the funnel. Thecombined mixture was extracted with hexanes three times (100 ml×3). Thethree hexane extracts were combined and dried over MgSO₄, filtered, andconcentrated to give the optically active butyrate 24 (1.12 g, 5.48mmol, 40%). Enantiomeric excess was determined by an NMR experimentusing aTris[3-heptafluoropropyl-hydroxymethylene)-(+)-camphorato]europium (III)derivative as a chemical shift reagent; this procedure showedapproximately 40% enrichment for one enantiomer. The remaining aqueouslayer from the reaction was subjected to a continuous extraction withCH₂ Cl₂ for 20 hours. The organic layer was removed from the extractionapparatus, dried over MgSO₄, filtered, and concentrated to give an oil(1.24 g), which was shown by NMR analysis to consist of predominatelythe 2-hydroxymethyl-5-oxo-1,3-oxathiolane 25 with small amounts ofbutyric acid and the butyrate 24.

The lactone 25 (0.85 g, 4.16 mmol) was dissolved in toluene (30 ml), andthe solution cooled to -78° C. Dibal-H solution (9 ml, 1.0 M in hexanes,9 mmol) was added dropwise, while the inside temperature was kept below-70° C. throughout the addition. After the addition was completed, themixture was stirred for 0.5 hours at -78° C. Acetic anhydride (5 ml, 53mmol) was added and the mixture, with continuous stirring, was allowedto reach room temperature overnight. Water (5 ml) was added to thereaction mixture and the resultant mixture was stirred for 1 hour. MgSO₄(40 g) was then added and the mixture was stirred vigorously for 1 hourat room temperature. The mixture was filtered, concentrated, and theresidue flash chromatographed with 20% EtOAc in hexanes to give acolorless liquid 26 (0.41 g, 1.86 mmol, 45%) which was a mixture ofanomers at the C-4 position.

The 2-Acetoxymethyl-5-acetoxy-1,3-oxathiolane 26 (0.40 g, 1.82 mmol) wasdissolved in 1,2-dichloroethane (40 ml), and to it the silylatedcytosine 12 (0.70 g, 2.74 mmol) was added, all at once, at roomtemperature. The mixture was stirred for 10 minutes, and to it a SnCl₄solution (3.0 ml, 1.0 M solution in CH₂ Cl₂, 3.0 mmol) was added,dropwise, at room temperature. Additional SnCl₄ solution (1.0 ml) wasadded after 1 hour. The reaction was followed by TLC. Upon completion ofthe coupling, the solution was concentrated, the residue was trituratedwith triethylamine (2 ml) and subjected to flash chromatography (firstwith neat EtOAc then 20% ethanol in EtOAc) to give a tan solid 27 (0.42g, 1.55 mmol, 86%). (¹ H NMR: 7.73 (1H, d, H₆) J=7.5 Hz; 6.33 (1H, t,H₄,) J=4.8 Hz; 5.80 (1H, d, H₅) J=7.5 Hz; 4.52 (1H, dd, 1H₅,) J=5.7 and12.3 Hz; 4.37 (1H, dd, 1H₅,) J=3.3 and 12.3 Hz; 3.54 (1H, dd, H₂,) J=5.4and 12.0 Hz; 3.10 (1H, dd, 1H₃); 2.11 (3H, s, CH₃))

The 5'-Acetate of BCH-189 27 (140 mg. 0.52 mmol) was dissolved inanhydrous methanol (10 ml), and to it was added sodium methoxide (110mg, 2.0 mmol) in one portion. The mixture was stirred at roomtemperature until the hydrolysis was complete. The hydrolysis took about1 hour, and the reaction was followed by TLC. Upon completion, themixture was then concentrated, and the residue taken up with ethanol (2ml). The ethanol solution was subjected to column chromatography usingethyl acetate first, then 20% ethanol in EtOAc to afford a white foam(110 mg, 92%), which exhibited an NMR spectrum identical to that ofauthentic BCH-189, 14.

What is claimed is:
 1. The (-)-enantiomer of the β-isomer of2',3'-dideoxy-3'-thia-cytidine (BCH-189).
 2. The (-)-enantiomer of theβ-isomer of 2',3'-dideoxy-3'-thia-cytidine (BCH-189), in substantiallypure form.
 3. The (-)-enantiomer of the β-isomer of2',3'-dideoxy-3'-thia-cytidine substantially free of the (+)-enantiomerof the β-isomer of 2',3'-dideoxy-3'-thia-cytidine.
 4. A compositionconsisting essentially of the (-)-enantiomer of the β-isomer of2',3'-dideoxy-3'-thia-cytidine.
 5. β-BCH-189 substantially in the formof one optical isomer.
 6. A composition consisting essentially of oneoptical isomer of β-BCH-189.
 7. Enantiomerically enriched β-BCH-189.